A Selection of Editing
By Jason Socrates
Bardi
Several years ago, Professor Norman Klinman of The Scripps
Research Institute was surprised when he received a review
copy of an article by a young immunologist he had met in Europe
in the early 1980s.
This immunologist, David Nemazee, who was then working at
the National Jewish Hospital in Denver, was making a bold
assertion that seemed to violate the theory of clonal selection—a
physiological process whereby the immune system generates
a diversity of B and T cells.
The clonal selection theory had been around since the 1950s,
when it was first suggested by the Australian immunologist
Frank Macfarlane Burnet. Nemazee's paper did not suggest that
clonal selection was wrong, but it was a significant departure
from that theory, which was subscribed to by most immunologists
of the time.
Clonal selection held that autoreactive B cells were merely
eliminated. But in the paper, Nemazee showed that B cells
have a proofreading quality-control mechanism enabling autoreactive
cells to edit, modify, and reexpress the receptor to become
non-autoreactive.
He called this mechanism receptor editing.
"[The B cells] seem to throw out part of the receptor and
replace it, keeping the other half," says Nemazee. "The body
can recycle cells that otherwise would be thrown out—we
thought it was quite important."
Such a stunning claim caused Klinman to raise an eyebrow.
He sent the article back to Nemazee, asking him to provide
more experimental evidence.
Nemazee did the experiments Klinman suggested, and published
the paper in 1993. Another group at Princeton University published
a similar finding at the same time.
"It was huge," says Klinman.
Making the Most of Recombination
Nemazee, who is now a professor at Scripps Research, has
always been interested in understanding tolerance (why antibodies
don't attack the body's own tissues). His discovery of receptor
editing came out of that interest.
The immune system faces a daunting problem in a world full
of pathogens: how can it possibly recognize the myriad viral
and bacterial antigens—pieces of protein or carbohydrate
that stimulate an immune response? Harder still, how can the
immune system anticipate new antigens from mutated viruses
and bacteria that don't even exist yet?
What enables a great number of foreign antigens to be recognized
by the immune system is the extraordinarily large T and B
cell "repertoire" that the body produces and maintains. This
diverse repertoire is generated within the body by a process
that involves the rearrangement of specific genes within these
B and T cells.
When a B cell develops from stem cells in the bone marrow,
a process that occurs continuously throughout life, it rearranges
its immunoglobin gene—that codes for both the large receptor
protein that sits on the surface of the B cell to recognize
antigen and the antibody that is specific for this antigen
once it is recognized.
Immunoglobins and antigens have two components, a heavy
chain and a light chain, which are recombined to develop an
incredible diversity of antibodies.
The rearrangement of the heavy chain occurs first, and it
involves bringing together three segments (termed V, D, and
J for variable, joining, and diversity, respectively), which
are spliced together in a process that is appropriately named
V(D)J recombination. Since there are multiple copies of the
V, D, and J genes in the human genome, a recombined heavy
chain has over a million possible combinations.
Following recombination, the final sequence is permanently
spliced together as the heavy chain so that a mature B cell
will produce only one specific antibody. Then this "pro" B
cell is checked with a quality control mechanism to ensure
that the heavy chain is able to express, fold, and bind to
its eventual light chain. Most B cells will not make this
cut.
The ones that do will proliferate through a number of cell
divisions (the exact number is not known in humans) in a process
known as clonal expansion and become "pre" B cells.
"Cells at that stage are developmentally arrested," says
Nemazee. "They are not allowed to leave until they are fully
schooled."
The schooling first involves a test. The immunoglobin gene
is checked to make sure that its immunoglobin receptor does
not recognize self antigen, which would lead the mature B
cell to produce autoreactive B cells. The B cells that are
not autoreactive at this point pass the test and are positively
selected and become immature B cells.
In the early 1980s, Nemazee made an in vivo model
to study tolerance. The model had only autoreactive B cells,
and Nemazee and colleagues tested what would happen if they
introduced the autoreactive cells to the models both in the
bone marrow and in the periphery.
"At that time, says Nemazee, "the thinking was that the
[autoreactive B cells] should have all been eliminated."
However, they noticed some of the B cells escaped elimination
when they were introduced into the bone marrow. Upon closer
inspection, they were startled to find that these no longer
expressed an autoreactive receptor. The receptor had been
"edited." This was the work Nemazee submitted for publication
that Klinman reviewed.
Receptor editing allows the immune system to correct a bad
receptor rather than throwing the whole cell away. Through
editing, the immune system saves time and energy, getting
the most antibody diversity at the lowest cost.
Once B cells are committed to their receptor, they leave
the bone marrow and circulate through the blood, lymph nodes,
and spleen, where they mature further. When they bind to the
antigen directly or are activated by other components of the
immune system, they begin the process of proliferating and
secreting antibodies to mark bacteria and viruses for destruction.
Application to Disease
Now Nemazee is interested in basic questions of biochemistry
and molecular biology related to receptor editing, such as
how recognition of self antigen turns the genes that control
recombination on and off.
He is also studying what regulates the survival of B cells,
looking for the genes that are involved in B cell survival.
"Your bloodstream is full of B cells that have probably been
around for years and have never divided since they were produced,"
Nemazee says. There is very little data showing which genes
are involved in keeping non-autoreactive cells alive preferentially.
Nemazee is also interested in the implications of receptor
editing for human health. A number of diseases, such as lupus
and rheumatoid arthritis, are caused by B cells making antibodies
against self tissue. This sort of "friendly fire" leads to
a number of health problems.
"Maybe a defect in this process plays a role in autoimmune
diseases or in immune deficiency diseases," he says.
One hypothesis he is looking at is that if a person's immune
system cannot edit autoreactive immunoglobin receptors efficiently,
then immune deficiency results as the B cells that produce
these autoreactive receptors are eliminated. He is looking
at in vivo models that have defects in the genes that control
receptor editing to see if they have an abnormal level of
B cells.
Another project in Nemazee's laboratory involves a collaboration
with Scripps Research Professor Dennis Burton on a grant that
went into effect on April 1 and is funded by the National
Institute of Allergy and Infectious Diseases, one of the components
of the National Institutes of Health.
Improving HIV Vaccine
The idea is to come up with a way of improving the body's
response to an HIV envelope DNA vaccine.
"As my colleague Dennis Burton has taught us, most of the
antibodies that you raise against HIV are irrelevant because
they see the wrong parts of the HIV envelope protein," says
Nemazee. "One would like to have a vaccine that induces more
antibodies to the right part of the HIV envelope protein—the
CD4 binding site."
The solution that Nemazee and Burton are working on involves
fusing the HIV envelope protein with a second protein that
both stabilizes the normal structure of the envelope protein
and stimulates better antibody production.
The approach was inspired by work on a broadly neutralizing
antibody discovered in the bone marrow of a 31-year-old male
who had been HIV positive without symptoms for six years.
Designated b12, the antibody has a long finger-like region
on its surface that penetrates the surface of the main viral
glycoprotein gp120 on HIV and prevents it from causing disease.
A team of scientists at Scripps Research and at the Glycobiology
Institute at Oxford University in the United Kingdom elucidated
the structure of the antibody a few years ago. See http://www.scripps.edu/news/press/081001.html.
Since gp120 on HIV is a protein that forms a trimer, vaccines
that mimic this structure may work better.
The protein to which they are fusing gp120 is called BAFF,
which is a B cell survival factor. Significantly, this may
help the antibody because BAFF naturally causes a huge increase
in antibody production when it is overexpressed.
Continuing the Tradition
Klinman helped the Department of Immunology at Scripps Research
recruit Nemazee in 1998. Around the same time, immunologist
William Weigel, one of the founders of the institute, was
retiring. Weigel had pioneered research into the area of immune
tolerance by showing in the 1960s and 1970s that B and T cell
immune tolerance could be induced in early in vivo
models, and he laid the foundations of the field by exploring
the parameters of tolerance.
With Weigel retiring, Klinman wanted to recruit a faculty
member who would work in the same area of immunology and carry
the torch.
"This is a department that has been extremely strong in
the issues of autoimmunity and tolerance," says Klinman. "It's
our history—it's who we are."
Send comments to: jasonb@scripps.edu
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